This invention relates to recombinant, isolated and other synthetic DNA useful in male-sterility systems for plants. In particular, the invention relates to restorable male-sterility systems. Male-sterile plants are useful for the production of hybrid plants by sexual hybridisation.
Hybrid plants have the advantages of higher yield and better disease resistance than their parents, because of heterosis or hybrid vigour. Crop uniformity is another advantage of hybrid plants when the parents are extensively homozygous; this leads to improved crop management. Hybrid seed is therefore commercially important and sells at a premium price.
Producing a hybrid plant entails ensuring that the female parent does not self-fertilise. There have been many prior proposals, mechanical, chemical and genetic, for preventing self-pollination. Among the genetic methods is the use of anther-specific genes or their promoters to disrupt the normal production of pollen grains. An anther-specific promoter, for example, can be used to drive a "male-sterility DNA" at the appropriate time and in the right place. Male sterility DNAs include those coding for lytic enzymes, including those that lyse proteins, nucleic acids and carbohydrates. Glucanases are enzymes which break down carbohydrates.
In EP-A-0344029 (Plant Genetic Systems (PGS)) and WO-A-9211379 (Nickerson International Seed Company Limited) glucanase-coding DNA features among possible malesterility DNAs. Although many plant glucanases have been characterised and the genes cloned in some cases (eg defence-related "PR" glucanases), to date no glucanase with properties consistent with a role in microspore release has been reported. Microspore release is the process by which the immature microspores are liberated from a protective coat of .beta.(1,3) poly-glucan (callose) laid down by the microsporogenous cells before meiosis (Rowley, Grana Palynol., 2, 3-31 (1959); and Heslop-Harrison, Can. J. Bot. 46, 1185-1191(1968) and New Phytol., 67, 779-786 (1968)). The anther-expressed glucanase responsible for the dissolution of this callose coat is known as callase. Callase is synthesised by the cells of the tapetum and secreted into the locule. The appearance of the enzyme activity is developmentally regulated to coincide precisely with a specific stage of microspore development.
The basis of the use of a glucanase as a sterility DNA lies in the fact that mis-timing of the appearance of callase activity is associated with certain types of male-sterility (Warmke and Overman, J. Hered. 63 103-108 (1972)). Two types are recognised depending on whether the appearance of glucanase activity is premature or late. Since both types are found in nature, one important attraction of glucanase as a potential sterility DNA is that it already occurs in a natural system. Although plants that fail to produce active callase have not been described in nature, mutants of this type almost certainly occur. Failure to produce callase would prevent microspore-release, thereby causing pollen abortion and male-sterility. So, preventing callase expression would form the basis of a male-sterility system.
Several studies suggest that callase is probably different from other types of glucanases, such as the "PR" glucanases. For example, callase activity may be subject to both transcriptional and post-transcriptional control. This is suggested by the fact that there is a strong relationship between locule pH, callase activity, and the timing of microspore release (Izhar and Frankel, Theor. and Appl. Genet. 41, 104-108 (1971)). Locule pH and callase activity change coordinately in a developmentally regulated manner. In fertile Petunia hybrida anthers, the pH during meiosis is 6.8-7.0 and callase activity is undetectable. Following meiosis, at the tetrad stage, the locule pH drops in a precipitous fashion to 5.9-6.2 and callase activity increases sharply resulting in microspore release.
In certain male-sterile Petunia strains, the drop in pH and the appearance of callase activity are precocious and apparently result in the breakdown of microsporogenesis. Similarly, in another class of mutants, the drop in locule pH and the appearance of callase activity are both late and apparently result in the abortion of the microspores.
Thus, it appears that:
(1) the timing of the appearance of callase activity is critical for normal microspore development. (Presumably the abortion of prematurely released microspores indicates that they must reach a certain developmental stage before becoming capable of surviving without the protection of the callose coat); PA1 (2) the decrease in locule pH parallels the appearance of callase activity; and PA1 (3) the two events (production of callase activity and pH drop) are coordinately regulated in some manner. PA1 (1) glucanases, such as defence-related "PR" glucanases may not function efficiently under the conditions within the locule and may therefore not prove sufficiently useful as components of male sterility DNAs; PA1 (2) in the event that such glucanases are active within the locule, maximum naturalness, in terms of mimicking existing types of male-sterile plants, would nevertheless demand the use of the authentic callase gene. In this respect a male sterility system based on the use of a callase gene would be superior to any previously described system; and PA1 (3) systems based on preventing callase expression by destroying the callase mRNA using anti-callase mRNA, ribozymes or a callase anti-sense RNA require detailed knowledge of the nucleotide sequence of the callase mRNA.
The exact nature of the co-ordinate regulation of callase activity and pH is not known. The drop in pH may activate an otherwise fully functional enzyme (passive activation). Alternatively, the enzyme may be synthesised in an inactive form, rather like the zymogen of a protease, and activated as a consequence of some pH-dependent event such as the removal of an N-terminal or C-terminal addition (positive activation). The fact that callase, and possibly all glucanases, including PR-glucanases, has no detectable activity above pH 6.3, well below that encountered in the anther before microspore release may favour a passive activation theory.
However, since current assays for callase are crude and rely on the measurement of activity, it is impossible to say whether the enzyme is: i) produced before microspore release, but in a non-functional form for later activation; ii) synthesised in an active form but only at the precise time it is required; or iii) synthesised in advance in an active form, stored within the tapetal cells in some kind of vesicle, and released into the locule at microspore-release. The fact that pH drop and callase activity are so consistently correlated, even in cases where callase activity is found well before the normal time of microspore release, might indicate that the enzyme is synthesised in an inactive form in advance of its requirement and that the pH drop is in some way responsible for its activation. The alternative is that the drop in pH triggers the synthesis of callase in the tapetal cells. The important point is that, without knowing which is correct, it is impossible to predict whether the expression of glucanases that are not callase will produce male sterility.
The fact that callase appears different in certain respects from previously characterised glucanases has three important consequences: